Co-administration of a parvovirus and a cytokine for therapy of pancreatic cancer

ABSTRACT

The application relates to a combination of a parvovirus and a cytokine, preferably IFNγ, for use in treating pancreatic cancer (PDAC), in particular a terminal stage of this disease.

INCORPORATION BY REFERENCE

All documents cited or referenced herein (“herein cited documents”), andall documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

FIELD OF THE INVENTION

The present invention relates to a combination of a parvovirus and acytokine, preferably IFNγ, for use in treating pancreatic cancer, inparticular a terminal stage of this disease.

BACKGROUND OF THE INVENTION

Pancreatic cancer is an aggressive malignancy with one of the worstoutcomes among all cancers. For all stages combined, the 5-year relativesurvival rate is only 5% (Ahmedin J, Siegel R, Ward E, Hao Y, Xu J andThun M. Cancer Statistics 2009. CA Cancer J Clin 2009;59:225-49). Theradical surgery (Whipple's operation) is the only curative option inthis aggressive tumor but can be offered to less than 20% of pancreaticductal adenocarcinoma cancer (PDAC) patients. Chemotherapy can be usedas adjuvant to surgery or in advanced stage pancreatic cancer where, ina small group of patients, it offers real benefit in terms of survivaland quality of life (Katz M H, Fleming J B, Lee J E, Pisters P W.Current status of adjuvant therapy for pancreatic cancer. Oncologist2010;15:1205-13). Nevertheless, the therapeutic options for PDACpatients, especially these with peritoneal carcinosis, are lacking.

Novel virus-based anticancer therapies involve the use of viruses eitheras replicating oncolytic agents, or as recombinant vectors for genetransfer (Kirn D H, McCormick F. Replicating viruses as selective cancertherapeutics Mol Med Today 1996;2:519-527). The autonomous parvovirusesMVMp and H-1 belong to a group of small (˜5 kb) non-integratingsingle-stranded DNA viruses. Their oncotropic and oncotoxic propertiesmake them promising candidates for both types of applications (CornelisJ J, Haag A, Kornfeld C et al. Autonomous parvovirus vectors In:Cid-Arregui A, Garcia-Garranca A, eds Viral Vectors: Basic Science andGene Therapy Natick, M A: Eaton Publishing 2000;97-118). Recently itcould be demonstrated that applying H-1PV as mono-therapy or assecond-line treatment after gemcitabine chemotherapy, caused thereduction of tumor growth, prolonged the survival of rats bearingpre-established pancreatic tumors and led to the suppression ofmetastases (Angelova A L, Aprahamian M, Grekova S P, Hajri A, Leuchs B,Giese N A, et al. Improvement of gemcitabine-based therapy of pancreaticcarcinoma by means of oncolytic parvovirus H-1PV. Clin Cancer Res2009;15:511-9). Furthermore, it was found that immunological mechanismsare involved in the anticancer activity of H-1PV with a strongcorrelation between the therapeutic effect of the virus and IFN-γexpression in the draining lymph nodes of pancreatic tumors (Grekova S,Aprahamian M, Giese N, Schmitt S, Giese T, Falk C S, et al. Immune cellsparticipate in the oncosuppressive activity of parvovirus H-1PV and areactivated as a result of their abortive infection with this agent.Cancer Biol Ther 2011;10:1280-9).

Despite the impressive results achieved the anticancer efficacy of themost promising parvovirus candidates for human clinical applications(including H-1PV) needs to be improved, e.g., as regards the extensionof life span after diagnosis and as regards particular tumors likepancreatic tumors.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide means for animproved parvovirus-based therapy. According to the invention this mayachieved by the subject matters defined in the claims.

Oncolytic viruses with their capacity to specifically replicate in andkill tumor cells emerged as a novel class of cancer therapeutics. Ratoncolytic parvovirus (H-1PV) was used to treat different types of cancerin preclinical settings and was lately successfully combined withstandard gemcitabine chemotherapy in treating pancreatic ductaladenocarcinoma in rats (PDAC).

The experiments resulting in the present invention are based on an ideathat the therapeutic properties of H-1PV may be boosted with IFNγ forthe treatment of late incurable stages of PDAC like peritonealcarcinosis. Rats bearing established orthotopic pancreatic carcinomaswith peritoneal metastases were treated with a single intratumoral(i.t.) or intraperitoneal (i.p.) injection of 5×10⁸ plaque forming unitsof H-1PV with or without concomitant IFNy application. Intratumoralinjection proved to be more effective than the intraperitoneal route incontrolling the growth of both the primary pancreatic tumors andperitoneal carcinosis, accompanied by migration of virus from primary tometastatic deposits.

Concomitant i.p. treatment of H-1PV with recIFNy resulted in improvedtherapeutic effect yielding an extended animal survival, compared toi.p. treatment with H-1PV alone. IFNy application enhanced theH-1PV-induced peritoneal macrophage and splenocyte responses againsttumor cells while causing a significant reduction in the titers ofH1-PV-neutralising antibodies in ascitic fluid. Thus, IFNγco-application together with H-1PV might be considered as a noveltherapeutic option to improve the survival of PDAC patients withperitoneal carcinosis.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1: Impact of IFNy addition or depletion on H-1PV immunomodulatingactivity. (A) Macrophages were isolated from the peritoneal cavity offour groups (n=3) of tumor bearing Lewis rats treated either with PBS(mock) or with an intratumoral injection of 5×10⁸ pfu/rat of H-1PV(H-1PV IT) combined either with an antibody against IFNγ (H-1PVIT+aIFNy) or recombinant IFNγ (H-1PV IT+recIFNγ). Cells were plated in48-well plates at a density of 5×10⁵ cells per well and stimulated ornot with LPS. TNFα production in the supernatants was measured 24 hrslater by ELISA. Average values and standard deviations are shown. (B)Peritoneal macrophages (5×10⁵/well) from the same groups of rats werecocultured or not with 1×10⁵ HA-RPC cells at a 5:1 ratio in 48-wellplates and the release of interleukins −10 and −12 was measured byELISA. Mean cytokine ratios and standard deviations are presented. (C)Single cell suspensions of rat splenocytes were labeled with CFSE,plated in 24 well plates at 1×10⁶ cells/well and cocultured or not with2×10⁵ HA-RPC cells at a 5:1 ratio. 48 hrs later cells were harvested andprocessed for FACS analysis of proliferation. All data were median fromthree animals from triplicate wells. Differences were consideredsignificant at p values below 0.05.

FIG. 2: Therapeutic effects of H-1PV+IFNγ combination and virusdistribution. (A) Lewis rats (n=28) bearing simultaneously inducedorthotopic tumors and peritoneal metastases were divided into fourgroups (n=7) and either left untreated (Control), inoculated i.t. (H-1PVintratumoral) or i.p (H-1PV intraperitoneal) with H-1PV in the absenceor presence of recIFNy (H-1PV+IFNγ intraperitoneal). After the sacrificeof two animals 1 week after treatment, the survival of five rats fromeach group was followed up to six months after tumor induction whenanimals were sacrificed. Median values were considered significant at pvalues below 0.05. (B) Two animals per group were sacrificed 1 weekafter H-1PV and/or IFNγ treatments. Total RNA was extracted from visibletumors and metastases, converted to cDNA and subjected to RT-PCRs toevaluate the presence of H-1PV DNA/unspliced mRNA and b-actintranscripts, using respective primers. The abbreviations for the routeand type of treatment are indicated on the figure. The source of thematerial (Tumour or Metastasis) is indicated in superscript.

FIG. 3: Macrophage activation after H-1PV+IFNγ combined treatment.Macrophages were isolated from the peritoneal cavity of the H-1PV andH-1PV+IFNγ intraperitoneally injected rats, plated at 5×10⁵/well in 48well plates and cocultured or not with 1×10⁵ HA-RPC cells. The ratios ofTNFα and IL-10 in supernatants were determined 24 hrs later by ELISA.

FIG. 4: Influence of IFNγ application on the generation of virusneutralizing antibodies. (A) Serum and ascitic fluid were collected fromall groups of virus-treated rats (see FIG. 2) and the titers of virusneutralizing antibodies (αH-1PV) were determined using cytotoxicityprotection assay on NB324K cells. The titers are expressed as thepercentage of antivirus protection offered by serum or ascitis dilutionscompared to mock infected cells. (B) Two groups of metastasis bearingrats were treated with two intraperitoneal injections of H-1PV (3×10⁸pfu/injection per animal) spanning four weeks between them, with orwithout intermediate recIFNγ i.p. inoculation. Titers of αH-1PV inascitic fluids were determined 10-30 days after the second H-1PV i.p.injection and expressed as indicated above.

FIG. 5: Release of TNFα from PDAC and PBMC cocultures after H-1PV+IFNγtreatment. The indicated pancreatic cancer cell lines were seeded into10 cm² dishes at 1.5×10⁶ cells/dish and infected or not with H-1PV at anMOI of 10 pfu/cell. 24 hpi cells were harvested and plated ontopre-isolated PBMCs in 48 well plates at a ratio of PDAC:PBMC 1:5. Thecocultures were treated or not with 50 UI/ml of human recombinant IFNγand the release of TNFα was measured in supernatants 24 hrs later byELISA. Mock or H-1PV infected (MOI 10) monocultures of PBMCs served ascontrols. The indicated values are average of at least three independentexperiments. SD values are shown.

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention provides a combination of a parvovirus and acytokine, preferably a parvovirus and a cytokine as separate entities,e.g. in separate containers, for use in treating pancreatic cancer.

This combination of compounds is particularly useful for treating aterminal stage of pancreatic cancer. “Terminal stage” means a diseasethat cannot be cured or adequately treated and that is reasonablyexpected to result in the death of the patient within a relatively shortperiod of time, e.g. within some weeks or months. The combination ofcompounds is suitable for treating in particular an incurable stage,like peritoneal carcinosis. Peritoneal carcinosis represents theadvanced evolutive stage of several tumors that develop into abdominalorgans, such as colon, ovary, appendix, stomach, pancreas and liver.When the disease increases, the tumoral cells reach and affect themembrane covering the same organs (visceral peritoneum). Once this“barrier” has been passed, the affected cells are able to move into theabdominal cavity, carried by the peritoneal fluid. Even in mesotheliomacases that affect directly the peritoneum, tumoral cells can break offthe membrane and fall into the peritoneal fluid. The tumoral cellspresent into the liquid can die or survive feeding on substancescontained in the same liquid. These cells tend to accumulate in thosepoints of greater liquid readsorption, creating agglomerates that growmore and more, spreading into the whole abdomen and originating thecarcinosis.

The term “parvovirus” as used herein comprises wild-type or modifiedreplication-competent derivatives thereof, as well as related viruses orvectors based on such viruses or derivatives. Suitable parvoviruses,derivatives, etc. as well as cells which can be used for activelyproducing said parvoviruses and which are useful for therapy, arereadily determinable within the skill of the art based on the disclosureherein, without undue empirical effort. Rodent parvoviruses arepreferred. Particularly preferred are the following rodent parvoviruses:H1 (H1-PV), LuIII, Mouse minute virus (MMV), Mouse parvovirus (MPV), Ratminute virus (RMV), Rat parvovirus (RPV) and Rat virus (RV).

Patients treatable by the combination of agents according to theinvention include humans as well as non-human animals. Examples of thelatter include, without limitation, animals such as cows, sheep, pigs,horses, dogs, and cats.

As used herein, the term “cytokine” relates to a category of signallingmolecules that are used extensively in cellular communication. Theycomprise proteins, peptides, or glycoproteins. The term cytokineencompasses a large family of polypeptide regulators that are producedwidely throughout the body by cells of diverse embryological origin. Theaction of cytokines may be autocrine, paracrine, and endocrine. Allcytokines are critical to the development and functioning of both theinnate and adaptive immune response. They are often secreted by immunecells that have encountered a pathogen, thereby activating andrecruiting further immune cells to increase the system's response to thepathogen. Relying on the assays shown in Examples 2 to 5 the personskilled in the art is in a position to select cytokines that showbeneficial effects when administrated according to the presentinvention.

Preferably, the cytokine of the present invention is an interferon. Allinterferons (IFNs) are natural cell-signalling proteins produced by thecells of the immune system of most vertebrates in response to challengessuch as viruses, parasites and tumor cells. Interferons are produced bya wide variety of cells in response to the presence of double-strandedRNA, a key indicator of viral infection. Interferons assist the immuneresponse by inhibiting viral replication within host cells, activatingnatural killer cells and macrophages, increasing antigen presentation tolymphocytes, and inducing the resistance of host cells to viralinfection. All interferons in general have several effects in commonand, accordingly, the results obtained by use of IFN-γ in combinationwith a parvovirus, preferably H1-PV, might apply to further interferons.Interferons are antiviral and possess antioncogenic properties,macrophage and natural killer cell activation, and enhancement of majorhistocompatibility complex glycoprotein classes I and II, and thuspresentation of foreign (microbial) peptides to T cells. The productionof interferons is induced in response to microbes such as viruses andbacteria and their products (viral glycoproteins, viral RNA, bacterialendotoxin, bacterial flagella, CpG sites), as well as mitogens and othercytokines, for example interleukin 1, interleukin 2, interleukin-12,tumor necrosis factor and colony-stimulating factor, that aresynthesised in the response to the appearance of various antigens in thebody. Their metabolism and excretion take place mainly in the liver andkidneys. They rarely pass the placenta but they can cross theblood-brain barrier.

There are three major classes of interferons that have been describedfor humans:

(a) Interferon type I: The type I interferons present in humans areIFN-α, IFN-β and IFN-ω.

(b) Interferon type II: In humans this is IFN-γ.

(c) Interferon type III: Signal through a receptor complex consisting ofIL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12).

In a preferred embodiment of the present invention, the interferon isinterferon-γ (IFNγ).

Preferably, for the therapeutic use of the present invention theparvovirus and the cytokine are present in an effective dose andcombined with a pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable” is meant to encompass any carrier, which does not interferewith the effectiveness of the biological activity of the activeingredients and that is not toxic to the patient to whom it isadministered. Examples of suitable pharmaceutical carriers are wellknown in the art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Such carriers can be formulated by conventionalmethods and can be administered to the subject at an effective dose.

An “effective dose” refers to amounts of the active ingredients that aresufficient to affect the course and the severity of the tumor, leadingto the reduction or remission of such pathology. An “effective dose”useful for treating and/or preventing these diseases may be determinedusing methods known to one skilled in the art (see for example, Fingl etal., The Pharmocological Basis of Therapeutics, Goodman and Gilman, eds.Macmillan Publishing Co., New York, pp. 1-46 ((1975)).

Preferred doses of the parvovirus are in the range of about 10⁸ to 10⁹pfu (single injection) in rats and of the cytokine, in particular IFNy,in the range of about 10⁵ to 10⁶ IU (single injection). For humans thepreferred effective dose of the parvovirus is approximately 10¹¹ pfu andof the cytokine (e.g. IFNγ) about 2×10⁶ to 10⁸IU.

Additional pharmaceutically compatible carriers can include gels,bioasorbable matrix materials, implantation elements containing thetherapeutic agent, or any other suitable vehicle, delivery or dispensingmeans or material(s).

Administration of the compounds may be effected by different ways, e.g.by intravenous, intraperetoneal, subcutaneous, intramuscular, topical orintradermal administration. The route of administration, of course,depends on the kind of therapy and the kind of compounds contained inthe pharmaceutical composition. The dosage regimen of the parvovirus andthe cytokine is readily determinable within the skill of the art, by theattending physician based on patient data, observations and otherclinical factors, including for example the patient's size, body surfacearea, age, sex, the particular parvovirus to be administered, the timeand route of administration, the tumor type and characteristics, generalhealth of the patient, and other drug therapies to which the patient isbeing subjected.

If the parvovirus comprises infectious virus particles with the abilityto penetrate through the blood-brain barrier, treatment can be performedor at least initiated by intravenous injection of the parvovirus, e.g.,H1 virus. As another specific administration technique, the parvovirus(virus, vector and/or cell agent) containing composition can beadministered to the patient from a source implanted in the patient. Forexample, a catheter, e.g., of silicone or other biocompatible material,can be connected to a small subcutaneous reservoir (Rickham reservoir)installed in the patient during tumor removal or by a separateprocedure, to permit the parvovirus containing composition to beinjected locally at various times without further surgical intervention.The parvovirus or derived vectors containing composition can also beinjected into the tumor by stereotactic surgical techniques or byneuronavigation targeting techniques. Administration of the parvoviruscontaining compositions can also be performed by continuous infusion ofviral particles or fluids containing viral particles through implantedcatheters at low flow rates using suitable pump systems, e.g.,peristaltic infusion pumps or convection enhanced delivery (CED) pumps.

As yet another method of administration of the parvovirus containingcomposition is from an implanted article constructed and arranged todispense the parvovirus containing composition to the desired cancertissue. For example, wafers can be employed that have been impregnatedwith the parvovirus containing composition, e.g., parvovirus H1, whereinthe wafer is attached to the edges of the resection cavity at theconclusion of surgical tumor removal. Multiple wafers can be employed insuch therapeutic intervention. Cells that actively produce theparvovirus, e.g., parvovirus H1, or H1 vectors, can be injected into thetumor, or into the tumoral cavity after tumor removal.

Preferably, the parvovirus and the cytokine are administered as separatecompounds. The administration of the cytokine, when administeredseparately, can be accomplished in a variety of ways. A preferred routeof administration of the parvovirus is intratumoral administration. Apreferred route of administration of the cytokine is intraperitonealadministration. The combination of both routes of administration showssynergistic effects.

The therapeutic efficacy of the combination of compounds according tothe present invention can be further improved by co-administration of animmunosuppressive agent like rapamycin or cyclophosphamide.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1 Materials and Methods

(A) Cells and reagents. Human pancreatic carcinoma cell lines fromprimary (Panc-1, MiaPaCa-2, B×PC-3) or metastatic (Capan 1, T3M4,AsPC-1, Colo357) tumors, were obtained from ATCC (Manassas, Va.) andgrown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS).The HA-RPC cell line (ATCC, LGC Standards, Wesel Germany) derived from achemically induced pancreatic ductal adenocarcinoma in Lewis rats wasgrown in DMEM supplemented with 10% FCS. Human NB324K cells (ATCC, LGCStandards, Wesel Germany) used for cytotoxicity protection assays werecultured in MEM medium with 5% FCS. All media were supplemented withpenicillin (100 U/ml) and streptomycin (100 μg/ml). Lyophilizedrecombinant rat and human IFNy were obtained from Biomol GMBH (Hamburg,Germany) and reconstituted in sterile deionized water. The mousemonoclonal antibody clone DB-1 with specificity against murine IFNγ(αIFNγ) was produced in bulk amount by NatuTec GmbH (Frankfurt,Germany). Where indicated, in some experiments cells were stimulatedusing LPS at final concentration of 5 μg/ml.

For the isolation of peritoneal macrophages rats received an i.p.injection of 4 ml of 4% Thioglycolate solution in PBS three days beforesacrifice. After sacrificing the animals, 40 ml of sterile PBS wereinstilated in the peritoneal cavity and recovered using a syringe. Thecells were collected by centrifugation and plated in DMEM containing 10%FCS and antibiotics.

Peripheral blood mononuclear cells (PBMC) were isolated from theheparinized blood of randomly selected healthy donors by differentialcentrifugation over Histopaque (Sigma) and cultured in RPMI with 10% FCSand antibiotics. Peripheral blood macrophages were enriched by adherenceto plastic surface. Buffy coats were obtained from the blood bank ofIKTZ Heidelberg.

(B) Virus-neutralizing antibody detection. Serial dilutions of the seraof experimental animals were made in MEM and mixed with an equal volumeof H-1PV virus suspension (corresponding to 2×10⁴ pfu/well). Afterincubation for 30 min. at 37° C., the mixture was inoculated onto NB324Kcells plated in 96-well plates (2×10³ cells/well). The cell survivalrates were assessed after 72 h using a MTT(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay.

(C) Semi-quantitative RT-PCR. For RT-PCR total RNA was extracted frompancreatic tumors or metastatic nodules of treated animals, reversetranscribed into cDNA, and PCRs for H-1PV and B-actin were performedusing previously described primer sequences and conditions (Grekova S,Aprahamian M, Giese N, Schmitt S, Giese T, Falk C S, et al. Immune cellsparticipate in the oncosuppressive activity of parvovirus H-1PV and areactivated as a result of their abortive infection with this agent.Cancer Biol Ther 2011;10:1280-9).

(D) ELISA. Measurement of rat TNFγ; IL-10, IL-12 and human TNFα releasewas done using commercially available ELISA kits from eBioscience(Frankfurt, Germany) as described by manufacturer.

(E) FACS determination of splenocytes' proliferation index. Rat spleenswere pressed against a mesh to obtain single cell suspensions andsplenocytes were adjusted to a concentration of 5×10⁶/ml in PBS. Thestock 5 mM CFSE solution was diluted at 1/1000 in PBS (a finalconcentration of 5 μM), added to lymphocytes pellet and mixed rapidly.After incubation for 5 minutes at room temperature 10 volumes of PBScontaining 5% FSC were added and the cells were centrifuged. Washes inPBS/FCS were repeated 3 times. Labeled splenocytes were co-cultured withHA-RPC cells or alone as a control. After 72 h of incubation, cells werecollected, washed and measured for CFSE fluorescence using FACSCalibur(BD, California, USA). The proliferation index was calculated based onthe level of reduction in fluorescence intensity of the cultures.

(F) Animal studies. The orthotopic rat model using HA-RPC cells has beenpreviously described (5). For the induction of metastasis a cellsuspension was prepared in phosphate-buffered saline (PBS) out ofsubcutaneous tumors preformed by implantation of HA-RPC cells andinjected intraperitoneally to Lewis rats at 3×10⁶ cells in 500 μl peranimal.

Rat recIFNγ was injected in 3 consecutive weeks at 30 000 UI/week i.p.in a 100 μl volume for a total dose of 90 000 UI/animal. The aIFNyantibody was applied at the same times at 0.8 mg/animal for a total doseof 2.4 mg. Ascitic fluid was obtained using a peritoneal puncture underaerosol anesthesia at the time before animal sacrifice.

(G) Statistical methods. Means and standard deviations were calculatedfrom at least three animals in triplicate wells in vitro experiments.Statistical differences were assessed using Student's t test andWilcoxon test. For in vivo mortality data assessment, experimentalgroups were compared with log-rank test.

Example 2 RecIFNγ Contributes to the Immunomodulating Anticancer Effectof H-1PV

As a first step the potential impact of interferon-γ on theimmunomodulating features of parvovirus H-1PV in pancreatic cancer wasestablished. Therefore, H-1PV was applied in tumors raisedorthotopically through injection of HA-RPC cells in the pancreatic tailof three groups of rats using a PBS-treated group as control. Virustreatment was also combined either with intraperitoneal recombinant ratinterferon (recIFNγ) or with a neutralizing antibody against it (αIFNγ).Three days later animals were sacrificed to perform immunologicalprofiling of splenocytes and peritoneal macrophages. The cells werecultured for 48 hours either alone (no treat), together with HA-RPC ratpancreatic cancer cells (the cell line used for initiating the tumors)or LPS. Different parameters related to the anticancer immune response,like the production of TNFα (FIG. 1A), the IL-12/IL-10 ratio (FIG. 1B)of cytokines released by macrophages, as well as the proliferationcapacity of splenocytes (FIG. 1C) were analyzed. The supernatants ofmacrophages isolated from rats, in which H-1PV was combined with recIFNycontained up to 1 ng more TNFα, compared to those obtained from animalstreated with virus only. The lowest levels of TNFα release were measuredeither in the non-treated mock (0.2 and 0.5 ng) or when rats weretreated with a neutralizing antibody against interferon gamma (0.3 and 1ng). A similar pattern of effects of recIFNγ and αIFNγ was detected whencomparing the IL-12/IL-10 ratios of the different macrophage cultures.In addition, the combination of H-1PV with recombinant interferon gammacaused a significant twofold increase in the proliferative potential ofsplenocytes both spontaneously and in the presence of tumor cells.

These data pointed that H-1PV application alone can activate peritonealmacrophages or combined with recIFNy i.p. introduction could change theactivation status of immune cells both in spleen and in the peritonealcavity leading to predominance of immuno-stimulatory cytokines (TNFα,IL-12) over the immunosuppressive factors (IL-10). The decrease of theabove-mentioned immunological parameters upon depletion of IFNy,especially in the case of peritoneal macrophages, confirmed ourassumption that this cytokine plays a role in stimulating the innateimmune system as part of the immunomodulating effect of oncolytic H-1PV.

Example 3 RecIFNγ Improves the Therapeutic Potential of H-1PV for theTreatment of PDAC Peritoneal Carcinosis

Since the results obtained were encouraging a combination of recombinantIFNγ and H-1PV parvovirus was used for the treatment of one of the mostlethal complications of pancreatic cancer in humans, namely the spreadof the tumor to the peritoneal cavity. To mimic this situation tumorsboth in the pancreas and in the peritoneal cavity of Lewis rats wereinduced. Two weeks later, the rats were randomly divided into fourgroups, in which H-1PV was applied through two different routes(intratumoral or intraperitoneal). In one group the virus i.p.inoculation was combined with recIFNγ using the same route (FIG. 2Aprotocol). Animal survival was followed (FIG. 2A) confirming that H-1PVintratumoral injection was still most effective to protect rats againstPDAC with two animals remaining tumor free more than six months aftertreatment (5). H-1PV could significantly improve the survival of ratsupon peritoneal application compared to the control group but was stillless effective in comparison to the i.t. route. Notably, the combinationwith recIFNγ could significantly improve the effect of the virusextending the median survival from 83 to 96 days (FIG. 2A).

Two animals per group were sacrificed one week after treatment toanalyze virus presence by RT-PCR (FIG. 2B). The distribution of viralDNA signals showed that (i) the virus could migrate from the primarytumor after intratumoral application (HIT^(Tu)) to metastasis (HIT^(M))in the peritoneal cavity, (ii) it can infect metastasis uponintraperitoneal inoculation (HIP^(M)), and (iii) that upon i.p.combination with H-1PV IFNγ does not change significantly the viruslevels in metastases (compare HIP^(M) and HIFN^(M)).

Isolation of peritoneal macrophages from mock, H-1PV or H-1PV with IFNγintraperitoneally treated rats showed that the ratio between TNFα andIL-10 produced was significantly increased in the presence ofrecombinant IFNγ when macrophages were cocultured with HA-RPC cells,speaking in favor of phagocytes' activation (FIG. 3).

In conclusion, intratumoral application of H-1PV seems to have asuperior effect compared to intraperitoneal inoculation for thetreatment of PDAC. In case intraperitoneal inoculations of the virus areperformed at the stage of advanced metastatic disease, a combinationwith IFNy can be very favorable.

Example 4 RecIFNγ Cotreatment Reduces the Titers of H-1PV NeutralizingAntibodies in Ascitic Fluid

One of the major functions of IFNy is its ability to prime the cellular(through Th1 cells/cytokines) and to down-modulate the humoral (throughTh2 cells/cytokines) immune response. Therefore, it was assumed that thecombination of IFNγ and oncolytic H-1PV may also reduce the titers ofneutralizing antibodies produced against the virus. In order to addressthis hypothesis, serum from peripheral blood and ascitic fluid from theperitoneal cavity of rats participating in the above-mentionedexperiment were collected and the titers of αH-1PV antibodies weredetermined, using a cytotoxicity protection assay on virus-sensitivecells. It was found that in the first experiment performed, no evidentdifference could be detected in the titer of αH-1PV in animal serairrespective of the virus inoculation route and IFNγ treatment (FIG. 4Aupper pannel). Similarly, the inoculation route had no impact on theantiviral titers in ascitic fluid collected in the time-frame (20 to 40days) after virus treatment. On the other hand, co-application ofrecIFNγ together with H-1PV caused a significant reduction (from 1:5000to 1:1280) in the titers of αH-1PV in the ascitic fluid of the animalsmost probably due to the stronger effect of i.p. applied IFNγ.

Then, in a modified experimental setting it was tested whether theIFNγ-provoked drop in antiviral antibodies within ascites would increasethe levels of H-1PV DNA in metastases when this cytokine is appliedbefore a second virus inoculation. First, it was noticed that the titersof αH-1PV in ascitic fluid collected within 10 to 30 days after thissecond H-1PV i.p. injection (FIG. 4B) were much higher than the onesinduced by a single H-1PV i.p. application (FIG. 4A). This effect wasmost probably due to boosting of the immune system related to therepeated virus application. Interestingly, when IFNγ was applied beforethe second H-1PV inoculation (H−1+IFNγ) it was noticed that αH-1PVtiters remained similar to the ones observed in fluids from animalssubjected to a single virus inoculation (compare FIG. 4B with FIG. 4A,lower panel), suggesting that the cytokine has inhibited theoverproduction of αH-1PV triggered by the second virus injection.

The H-1PV transduction level of metastasis in the two groups of ratsafter the second virus application (SFIG. 1) was also evaluated.Unfortunately, IFNγ treatment had no positive impact on the amounts ofviral DNA in metastasis despite the reduction of antiviral antibodies(FIG. 4B) suggesting that this reduction was not sufficient to overcomethe antibody pressure in ascitic fluid.

Example 5 Rec IFNγ Can Improve the Effect of H-1PV to Stimulate theHuman Innate Immune System

In search of clinical relevance of the obtained data, the previousstudies were continued using human PDAC cell lines and peripheral bloodmonocytes derived from healthy donors, aiming to find out whether thelatter can be activated more efficiently with a combination of virus andIFNγ. It was previously reported that H-1PV infection leads to a limitedbut significant activation of human PBMCs as indicated by their TNFαrelease. The latter effect was largely masked in the case when PBMCswere cocultivated with pancreatic cancer cells irrespective of theirinfection status (Grekova S, Aprahamian M, Giese N, Schmitt S, Giese T,Falk C S, et al. Immune cells participate in the oncosuppressiveactivity of parvovirus H-1PV and are activated as a result of theirabortive infection with this agent. Cancer Biol Ther 2011;10:1280-9).Considering that PDAC cells can express IFNγ receptors first of all thelethal effect of H-1PV and IFNγ combination on pancreatic cancer cellwas evaluated. IFNγ does not change H-1PV-induced toxicity on human PDACcells (SFIG. 2). In a next step, PBMCs were cocultured with pancreaticcancer cells that had been previously infected (or not) with H-1PV, andused the release of TNFα as a read-out for innate immune cellactivation. As already previously reported, the direct infection ofPBMCs with H-1PV resulted in an increased release of TNFα at 48 hpi(FIG. 5, PBMC monoculture) (Grekova S, Aprahamian M, Giese N, Schmitt S,Giese T, Falk CS, et al. Immune cells participate in the oncosuppressiveactivity of parvovirus H-1PV and are activated as a result of theirabortive infection with this agent. Cancer Biol Ther 2011;10:1280-9).Addition of relatively low dose IFNy (50 UI/ml) to the cultures did notsignificantly enhance TNFα production. The same was the case when PDACswere pre-infected with H-1PV before coculturing them with the PBMCs.However, in general, in the presence of IFNγ, PBMC cocultures withPanc-1, T3M4, Capan-1 and especially Colo357 and AsPC-1 produced 100-150pg/ml more TNFγ, corresponding to a higher level of activation of innateimmune cells. Interestingly, despite the fact that the fluctuations ofTNFα were not statistically significant, a tendency could be observedthat PDAC cells deriving from metastatic (lymph node, liver orperitoneal) pancreatic cancer seemed to be more potent stimulators ofPBMCs in the presence of IFNy than the lines established from primaryPDAC tumors. In general, all these effects support the hypothesis thatconcomitant application of IFNy can be beneficial for the anticancervaccination effect of H-1PV especially in the treatment of advancedmetastatic disease.

Example 6 Conclusion

The observed reduction in the titers of virus neutralizing antibodiesinduced by IFNγ represents a very interesting phenomenon in the frame ofoncolytic virotherapy. It is in agreement with the changes observed inthe II-12/II-10 cytokine ratio secreted from macrophages pointing to ashift in the Th1/Th2 balance in the peritoneal cavity. Probably, anadditional modification of the IFNγ treatment protocol or itscombination with certain immunosuppressive agents, recently reported inoncolytic virotherapy may improve the described effect and reduce theantibodies to levels permitting repeated virus applications andmetastasis transduction (Lun XQ, Jang J H, Tang N, Deng H, Head R, BellJ C, et al. Efficacy of systemically administered oncolytic vacciniavirotherapy for malignant gliomas is enhanced by combination therapywith rapamycin or cyclophosphamide. Clin Cancer Res 2009;15:2777-88).

Treatment of peripheral blood mononuclear cells with H-1PV could primethe release of TNFα, a cytokine that represents one of the main productssecreted upon macrophage activation possessing also strong antitumorproperties. However, coculturing PBMCs with pancreatic cancer cell linesderiving from different organ locations caused a generalized increase inTNFα levels that seemed to almost completely mask the effect of H-1PVpre-infection of PDAC cells. IFNγ could serve as an additionalstimulator of TNFα production mostly in the case of cocultures betweenPBMCs and metastatic PDAC cancer lines. Notably, this effect was mostpronounced for AsPC-1, a cell line deriving from a clinical case ofperitoneal metastasis, therefore giving stronger credibility to theresults obtained in animal experiments with peritoneal carcinosis. Inconclusion, the combination of an oncolytic virus with a powerfulimunomodulating cytokine like IFNγ may represent a promising strategyfor cancer therapy. In view of the forthcoming clinical applications ofH-1PV as an oncolytic agent, a therapeutic protocol involvingco-treatment with the two modalities has potential to improve theoutcome in terminal stage patients with pancreatic cancer.

The invention is further described by the following numbered paragraphs:

-   1. A combination of a parvovirus and a cytokine for use in treating    pancreatic cancer.-   2. The combination of compounds according to paragraph 1 for the use    according to paragraph 1 characterized in that the use is for    treating a terminal stage of pancreatic cancer.-   3. The combination of compounds according to paragraph 1 for the use    according to paragraph 2 characterized in that the use is for    treating a terminal stage of pancreatic cancer characterized by    peritoneal carcinosis.-   4. The combination of compounds according to paragraph 1 for the use    according to any one of paragraphs 1 to 3 characterized in that said    cytokine is an interferon.-   5. The combination of compounds according to paragraph 4 for the use    according to any one of paragraphs 1 to 3 characterized in that said    interferon is IFN-γ.-   6. The combination of compounds according to any one of paragraphs 1    to 5 for the use according to any one of paragraphs 1 to 3    characterized in that said parvovirus is a rodent parvovirus.-   7. The combination of compounds according to paragraph 6 for the use    according to any one of paragraphs 1 to 3 characterized in that said    rodent parvovirus is LuIII, Mouse minute virus (MMV), Mouse    parvovirus (MPV), Rat minute virus (RMV), Rat parvovirus (RPV), Rat    virus (RV) or H1 (H1-PV).-   8. The combination of compounds according to any one of paragraphs 1    to 7 for the use according to any one of paragraphs 1 to 3    characterized in that said parvovirus is intratumorally administered    and the cytokine is intraperitoneally administered.-   9. The combination of compounds according to any one of paragraphs 1    to 7 for the use according to any one of paragraphs 1 to 8    characterized in that the combination of compounds further comprises    an immunosuppressive agent.-   10. The combination of compounds according to paragraph 9 for the    use according to any one of paragraphs 1 to 8 characterized in that    the immunosuppressive agent is rapamycin or cyclophosphamide.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention. 11.A combination of a parvovirus and a cytokine for use in treatingpancreatic cancer.
 12. The combination of compounds according to claim11 characterized in that the use is for treating a terminal stage ofpancreatic cancer.
 13. The combination of compounds according to claim11 characterized in that the use is for treating a terminal stage ofpancreatic cancer characterized by peritoneal carcinosis.
 14. Thecombination of compounds according to claim 11 characterized in thatsaid cytokine is an interferon.
 15. The combination of compoundsaccording to claim 14 characterized in that said interferon is IFN-γ.16. The combination of compounds according to claim 11 characterized inthat said parvovirus is a rodent parvovirus.
 17. The combination ofcompounds according to claim 16 characterized in that said rodentparvovirus is LuIII, Mouse minute virus (MMV), Mouse parvovirus (MPV),Rat minute virus (RMV), Rat parvovirus (RPV), Rat virus (RV) or H1(H1-PV).
 18. The combination of compounds according to claim 11characterized in that said parvovirus is intratumorally administered andthe cytokine is intraperitoneally administered.
 19. The combination ofcompounds according to claim 11 characterized in that the combination ofcompounds further comprises an immunosuppressive agent.
 20. Thecombination of compounds according to claim 19 characterized in that theimmunosuppressive agent is rapamycin or cyclophosphamide.
 21. A methodfor treating pancreatic cancer comprising administering an effectiveamount of a combination of a parvovirus and a cytokine
 22. The methodaccording to claim 21 wherein the pancreatic cancer is a terminal stageof pancreatic cancer.
 23. The method according to claim 22 wherein theterminal stage of pancreatic cancer is characterized by peritonealcarcinosis.
 24. The method according to claim 21 wherein the cytokine isan interferon.
 25. The method according to claim 24 wherein theinterferon is IFN-γ.
 26. The method according to claim 21 wherein theparvovirus is a rodent parvovirus.
 27. The method according to claim 26wherein the rodent parvovirus is LuIII, Mouse minute virus (MMV), Mouseparvovirus (MPV), Rat minute virus (RMV), Rat parvovirus (RPV), Ratvirus (RV) or H1 (H1-PV).
 28. The method according to claim 21 whereinthe parvovirus is intratumorally administered and the cytokine isintraperitoneally administered.
 29. The method according to claim 21wherein the combination of compounds further comprises animmunosuppressive agent.
 30. The method according to claim 29 whereinthe immunosuppressive agent is rapamycin or cyclophosphamide.